The goal of this proposed research is to utilize a unique cholesterol-requiring mutant of cultured mammalian cells (cell line: Chinese hamster ovary) to probe the structural and functional roles of cholesterol in the mammalian cell membrane, and to study the regulation of cholesterol biosynthesis at the molecular level. These problems will be classified into two areas, and will be approached as follows: a) The biological function of membrane cholesterol. 1) Studies of effects of supplementation with various lipids on both short-term and long-term growth of wild-type and mutant cells in serum-free and delipidated-serum media. 2) Analyses of adaptive changes in membrane lipids and membrane proteins in wild-type and mutant cells after long-term exposure to limiting exogenous cholesterol supplementation, and/or after long-term exposure to exogenous cholesterol-analog supplementation. 3) Studies on plasma membrane enzyme activities after endogenous replacement of cholesterol by lanosterol and dihydrolanosterol in the mutant cells. b) Regulation of cholesterol metabolism. 1) Comparative studies on induction of 3-hydroxy-3-methylglutaryl CoA reductase in wild-type and cholesterol-requiring mutant cells. 2) Studies on sterol ester formation in wild-type and cholesterol-requiring mutant cells grown under various conditions. Studies on acyl CoA: cholesterol acyltransferase (ACAT) activities. 3) Isolation of ACAT-deficient mutants in CHO cells.